Project 3 ? ?Germline-mediated intergenerational epigenetic inheritance of paternal epimutations induced by a high fat diet? Project Abstract/Summary: Abundant data from many labs has established that environmental exposures can predispose development of disease characteristics in an exposed male and also in that male?s offspring, even if the offspring are never, themselves, directly exposed to the original disruptive influence. In addition to chemical exposures, deleterious lifestyle choices such as consumption of an unhealthy diet, lack of exercise, smoking, etc., can predispose disruptions of the epigenome (epimutations) that can be subsequently propagated to many cells or organs in the exposed male?s body, including to his sperm. Once in the exposed male?s sperm, lifestyle-induced epimutations can then be transmitted to the male?s offspring on the basis of epigenetic inheritance, where they can predispose development of similar disease characteristics. Though this phenomenon has now been studied for >10 years, there remains very little information about the underlying molecular mechanisms. We propose a novel, comprehensive, mechanism-focused set of experiments to be conducted using a mouse model subjected to two effects mimicking deleterious lifestyle choices in humans ? i) consumption of a high-fat diet and ii) lack of a structured exercise regime. Specifically, we propose experiments designed to 1) identify the specific combination of epigenetic parameters involved in transmission of lifestyle-induced epimutations from sperm to the ensuing fetus, 2) reveal the extent to, and mechanisms by, which resulting epimutations in the F1 fetus are propagated to developing somatic and germ cell lineages and on into the immature and adult offspring, 3) determine the extent of intercellular homo- versus hetero-geneity of lifestyle- induced epimutations in spermatogenic cells of the sire and in relevant germ and somatic cell types in his offspring, 4) discern the mechanisms by which inherited epimutations contribute to dysregulated gene expression in tissues relevant to aberrant/disease phenotypes in the offspring, 5) elucidate dysregulated pathways responsible for defective or disease states among offspring of sires transmitting lifestyle-induced epimutations, and 6) determine the extent to which the incidence of all of these deleterious effects can be reduced by transition of males from an unhealthy to a healthy life style, including a normal diet and exercise. A broad range of analyses of epigenomic parameters is proposed as a means to investigate mechanisms underlying the etiology and paternal transmission of lifestyle-induced, intergenerational epimutations. Results of the proposed research will inform future efforts to prevent, diagnose, treat or reverse the deleterious epimutagenic effects of siring offspring while engaged in an unhealthy life style.